One of the most surprising realisations to emerge from metagenomics studies in the early ‘00s was that the population of viruses and phages in nature is about 10 times larger than the population of prokaryotic organisms. Thus, bacteria and archaea are under constant pressure to develop resistance methods against a population of viruses with extremely high turnover and evolution rates, in what has been described as an evolutionary “arms race”. A novel, adaptive and heritable immune system encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral or plasmid sequences which are then used to inactivate the corresponding invader element via an RNA interference mechanism. A number of CRISPR-associated (Cas) protein families are responsible for the maintenance, expansion and function of the CRISPR loci. This system can be classified in a number of types and subtypes that differ widely in their gene composition and mode of action. This thesis describes the biochemical characteristics of CRISPR-mediated defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci transcription and their subsequent maturation into small guide crRNA units by the processing endonuclease of the system (Cas6) is investigated. After this step, different pathways and effector proteins are involved in the recognition and silencing of DNA or RNA exogenous nucleic acids. This thesis reports the identification and purification of a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue of which has been found to recognise and cleave RNA targets in P. furiosus. The recognition and silencing of DNA targets in E. coli has been shown to involve a multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD nuclease. S. solfataricus encodes orthologues for the core proteins of this complex, and the formation and function of an archaeal CASCADE is investigated in this thesis.

<p>Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.</p> / Dissertation

By 2030, the global colorectal cancer burden is projected to approximately double. This highlights the immediate need to expand our understanding of the etiological origins of colorectal cancer, so that novel therapeutic strategies can be identified and validated. The putative tumor suppressor gene RNF20 encodes a histone H2B mono-ubiquitin ligase and has been found altered/mutated in colorectal and numerous other cancer types. Several studies suggest that RNF20, and by extension mono-ubiquitinated histone H2B (H2Bub1), play important roles in maintaining genome stability in human cells. Indeed, hypomorphic RNF20 expression and/or function have been shown to underlie several phenotypes consistent with genome instability, making aberrant RNF20 biology a potential driver in oncogenesis. Through an evolutionarily conserved trans-histone pathway, RNF20 and H2Bub1 have been shown to modulate downstream di-methylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Accordingly, understanding the biology associated with RNF20, H2Bub1, H3K4me2, and H3K79me2 is an essential preliminary step towards understanding the etiological origins of cancer-associated RNF20 alterations and identifying a novel therapeutic strategy to selectively kill RNF20-deficient cancers. In this thesis, I employ single-cell imaging, and multiple biochemical techniques to investigate the spatial and temporal patterning and characterize the biology of RNF20, H2Bub1, H3K4me2 and H3K79me2 throughout the cell cycle. In addition, I employ the CRISPR-Cas9 genome editing system to generate RNF20-deficient HCT116 cells. Finally, I employ synthetic lethal strategies to selectively kill RNF20-depleted cells. In conclusion, the research chapters contained within this thesis have characterized putative drivers in cancer (Chapter 3), generated a valuable research reagent for CRISPR-Cas9 ii genome editing experiments (Chapter 4), and identified a novel therapeutic strategy to selectively kill certain cancer cells (Chapter 5). This thesis has increased our understanding of the etiological origins of cancer and generated novel reagents and treatments strategies that after further validation and clinical studies, could be employed to reduce morbidity and mortality rates associated with cancer. / October 2016

In prokaryotes, CRISPR/Cas9 system provides molecular immunity to bacteria and archaea against invading phages, conjugative plasmids and nucleic acids. CRISPR/Cas9 system has been adapted for targeted genome editing across diverse eukaryotic species for a variety of applications in basic and applied research. In this dissertation, I propose to adapt the CRISPR/Cas9 system to function as molecular immunity machinery against plant DNA viruses. Therefore, to test whether the CRISPR/Cas9 system is portable to plants, I produced plants stably over-expressing Cas9 and sgRNAs against single or multiple DNA viruses in Nicotiana benthamiana (N. benthamiana) and tomato (Solanum lycopersicum) plants. sgRNAs targeting the Cas9 endonuclease against different coding and non-coding viral sequences were tested in virus- interference experiments. I explored the possibility of generating robust interference against single and multiple DNA viruses. Subsequently, I studied the possibility of virus evasion of the CRISPR/Cas9 machinery and evolution of the virus escapees. Finally, I produced N. benthamaiana and tomato plants stably expressing the CRISPR/Cas9 machinery for developing durable virus resistance. Furthermore, developing effective viral-interference system in plants will help to understand the molecular underpinning of virus biology and host-defense mechanisms against plant viruses. In conclusion, my research project attempted to establish the efficacy and extend the utility of CRISPR/Cas9 system for viral interference in plants which promise exciting applications including producing engineered plants resistant to multiple viral infection.

Integration and expression of exogenous genetic material – in particular, transgenes – into the genomes of model organisms, cell lines or patients is widely used for the creation of genetically modified experimental systems and gene therapy. However, loss of transgene expression due to silencing is still a major hurdle which remains to be overcome. Judicious selection of integration loci can help alleviate the risk of silencing; in recent years the ability to efficiently and specifically target transgene integration has been improved by the advent of site-specific nucleases (SSNs). SSNs can be used to generate double strand breaks (DSBs) in a targeted manner, which increases the efficiency of homologous recombination (HR) mediated transgene integration into predetermined loci. In this work I investigate four human genomic loci for their potential to act as transgene integration sites which will support robust long term expression: the adeno-associated virus (AAV) integration site 1 (AAVS1); the human homologue of the mouse Rosa26 locus (hROSA26); the inosine monophosphate dehydrogenase 2 (IMPDH2) gene and the eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) gene. I also investigate the potential of creating a novel drug-selectable transgene integration system at the IMPDH2 locus to allow for rapid and specific selection of correctly inserted transgenes. In addition to their ability to drive targeted transgene integration, SSNs can be harnessed to specifically disrupt gene function through indel formation following erroneous repair of the induced DSB. Using this strategy, I aimed to answer some important biological questions about eukaryotic translation elongation factor 1 alpha (eEF1A); eEF1A is responsible for providing aminoacylated tRNAs to the ribosome during the elongation phase of protein synthesis. Humans and other vertebrates express two isoforms, eEF1A1 and eEF1A2 (encoded by EEF1A1 and EEF1A2 respectively). During development eEF1A1 is replaced by eEF1A2 in some tissues. The reasons for this remain elusive, but one explanation may lie in the moonlighting functions of eEF1A1, which may not be shared by eEF1A2. Additionally, eEF1A2 can act as an oncogene, while there is no evidence that eEF1A1 is overexpressed in tumours. To begin to untangle these issues I targeted EEF1A1 using SSNs with the aim of making a cell line expressing only the eEF1A2 isoform. This work suggests that eEF1A1 may be essential even in the presence of eEF1A2, though further studies will be required to confirm this.

DNA sequence information is constantly threatened by damage. In the clinic, intentional DNA damage is often used to treat cancer. Cisplatin, a first-line chemotherapy used to treat millions of patients, functions specifically by generating physical links within DNA strands, blocking DNA replication, and killing dividing cells. To maintain genome integrity, organisms have evolved the capacity to repair, respond, or otherwise resist change to the DNA sequence through a network of genetically encoded DNA damage tolerance pathways. In chapter 1, I present advances in experimental design and current progress for a systems genetics approach, using Saccharomyces cerevisiae, to reveal relationships between cisplatin tolerance pathways. Additionally, recent efforts to sequence thousands of cancer genomes have revealed recurrent genetic changes that cause overexpression of specific cisplatin tolerance genes. In chapter 2, I present a submitted manuscript that models overexpression of an essential cisplatin tolerance gene. This study uses a systems genetics approach to reveal the genetic pathways that are essential for tolerating this perturbation, which ultimately led to mechanistic insights for this gene. Convenient genome engineering in Saccharomyces has made this organism an ideal model to develop systems genetics concepts and approaches. In chapter 3, I present a published manuscript that demonstrates a new approach to disrupting genes by making site-specific nonsense mutations. Importantly, this approach does not require cytotoxic double-strand DNA breaks and is applicable to many model organisms for disrupting almost any gene, which may advance systems genetics into new model organisms. Systems genetics provides a framework for determining how DNA damage tolerance pathways act together to maintain cellular fitness and genome integrity. Such insights may one day help clinicians predict which cancers will respond to treatment, potentially sparing patients from unnecessary chemotherapy.

Signal integration and transduction by cell-surface receptors is a complex, multi-layered process resulting in tight regulation of downstream mediators, which in turn elicit pre-defined native cellular responses. The modular architecture of transmembrane receptors provides a unique opportunity for engineering de novo sensor/effector circuits, enabling the development of custom cellular functions for research and therapeutic applications. The signal transduction module of most existing chimeric receptors consists of either native intracellular domains or effectors domains fused to non-programmable DNA binding proteins. Therefore, these receptors can only engage in natural signalling pathways or drive the expression of artificial, pre-integrated transgenes. By harnessing the programmability of a nuclease deficient CRISPR/Cas9 (dCas9) signal transduction module and leveraging the evolutionarily optimised ligand-sensing capacity of native receptors, I have created a novel class of dCas9-based synthetic receptors (dCas9-synR). I demonstrate that an optimised split dCas9-based core architecture and custom protease-based signal release mechanism can be standardised across multiple classes of extracellular domains to engineer receptor tyrosine kinase (RTK)-based and G-protein-coupled receptor (GPCR)-based chimeric receptors. dCas9-synRTK and dCas9-synGPCR integrate a broad variety of input signals (peptides, proteins, lipids, sugars) with highly specific and robust activation of any custom output transcriptional programme in an agonist dose-dependent manner. Finally, to showcase the therapeutic potential of dCas9-synRs, I used them to convert a pro-angiogenic signal into an anti-angiogenic response, deploy a chemokine/cytokine programme in response to tumour-enriched biomolecules, and induce insulin expression following glucose stimulation. The performance of dCas9-synRs and their unique versatility in redirecting the information flow makes them ideally suited to engineer designer cells capable of sensing specific disease markers and in turn drive various therapeutic programmes.

Mammalian neural stem cell (NSC) lines provide a useful experimental model for basic and applied research across stem cell and developmental biology, regenerative medicine and neuroscience. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with functional genetic analyses. However, targeted genetic manipulations have not been reported for mammalian NSC lines. Here, we deploy the CRISPR/Cas9 technology and demonstrate a variety of diverse targeted genetic modifications in both mouse and human NSC lines such as: targeted transgene insertion at safe harbour loci; biallelic knockout of neurodevelopmental genes; knock-in of epitope tags and fluorescent reporters; and engineering of glioma driver mutations at endogenous proto-oncogenes. Leveraging these new optimised methods, we explored gene editing to model the earliest stages of paediatric gliomagenesis in primary human NSCs. Our data indicate that oncogenic mutations in histone H3.3 play a role in NSC transformation and may operate through suppression of replication induced senescence. By comparing cellular responses of NSC cultures from different compartments of the developing brain, we also identify differences in susceptibility to distinct H3.3 mutations that mirror the disease etiology. Altogether, our findings indicate that CRISPR/Cas9-assisted genome editing in NSC lines is a versatile tool to explore gene function in CNS development and cancer biology. Our project resulted in the creation of valuable human cellular models of paediatric gliomagenesis, which will allow us to further our understanding of the disease and carry out cell based drug discovery projects.

Remarkable progress has been made in our ability to design and produce synthetic DNA binding domains (TALE or Cas9-based), which can be further functionalized into synthetic transcription factors (sTFs). This technology is revolutionizing our ability to modulate expression of endogenous mammalian genes. Forced expression of cDNAs encoding transcription factors (TFs) is widely used to drive lineage conversions. However, this process is often inefficient and unreliable. Multiplex delivery of sTFs pool to activate endogenous master regulators and extinguish the expression profile of the host cell type could be a potential solution to this problem. We have developed a novel, simple TALE assembly method that enabled us to produce and screen large numbers of TAL effectors and compare their activity to dCas9-based TFs. During this process, we constructed many new functionally validated sTFs. Our ultimate goal is to test whether combining synthetic transcriptional activators and repressors can efficiently reprogram fibroblasts to NS cells or alternatively ‘program’ NS cell differentiation to neurons. We performed analyses of the transcriptome and chromatin accessibility of both fibroblasts and neural stem cells to unravel their core TF networks and their epigenetic state. This will allow us in the future the targeted design of sTFs and synthetic chromatin modifiers for specifically changing cell identity.

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.